The emergence of extensively drug-resistant tuberculosis (XDR-TB) poses a growing threat to national and international biosecurity and to TB control achievements. Current standard indirect drug susceptibility testing (DST) takes up to 3 months to identify XDR-TB, undermining efforts to control XDR-TB. The University of California at San Diego has assembled an international team of TB experts from academia, public health, and industry to advance the development and evaluation of 3 rapid tests (i.e., Main strip, molecular beacon, microscopic-observation drug-susceptibility assays) to detect resistance to 6 drugs defining XDR-TB (i.e., isoniazid, rifampin, ofloxacin, amikacin, kanamycin, and capreomycin) directly from sputa. The specific aims are (1) to reduce the average XDR-TB detection time from months to a week, (2) to demonstrate agreement between the rapid tests and standard DST, (3) to identify the genetic basis of discordant results in Aim 2, (4) to characterize global XDR-TB strains and the epidemiology of drug resistance, and (5) to study cost- effectiveness. In Year 1, creation of new probes and assay procedures for the rapid tests (Version-1) will be based on published information, publicly-available genomic databases and new information from genotypic analysis of existing XDR and drug-susceptible strains from four study sites (i.e., South Africa, Moldova, India, the Philippines). These study sites will enroll 2,475 TB patients in Years 2-4; collect clinical data, sputa, and culture isolates; and perform the three rapid tests and DST onsite. Strains identified as drug resistant by the DST but not by the rapid tests will be further examined through sequencing target genes, neighboring genes, or whole genomes to find new regions or points of difference compared to drug-susceptible strains. New mutations discovered through this process will inform creation of probes for Version-2 rapid tests, as needed, to be tested onsite along with Version-1. This 5-year study will result in novel tests to rapidly detect XDR-TB in a broad range of laboratory settings as well as a repository of diverse well-characterized TB strains for future studies. The long-term implication is the development of technologies and methods that can be adapted to rapidly detect new drug-resistant TB strains and to test for resistance to other anti-TB drugs. RELEVANCE (See instructions): XDR-TB is associated with high fatality rates, is highly communicable, and has the potential to cause massive disruption to public-health systems and economies at a local, national and global scale if not detected and treated rapidly. The study proposes to develop and evaluate three novel rapid tests to reduce XDR-TB detection time to better guide public health measures and patient management decisions.